For the treatment of tumors or the diagnosis and treatment of inflammations, for example, osteoarthritis, and skin diseases, many studies have been conducted on proteins or small materials, which are present specifically in disease foci. Thus, many kinds of such materials have been identified while studies on treatment with such materials have also been actively conducted. For example, it was shown that prostate-specific antigen (PSA) is frequently present in prostate cancer, and matrix metalloprotease (MMP) is highly expressed specifically in arthritis tissue or other tumor tissues compared to that in normal tissue. Thus, such materials have been targeted in disease research and treatment. However, if materials that are used for the diagnosis and treatment of diseases do not act specifically against such targets only, problems of side effects and low image quality will be caused by non-specific distribution of the materials. For this reason, there has been a demand for the development of formulations that remain or act specifically in their targets.
Meanwhile, only some small materials can enter the cytoplasm or nucleus of live cells through the cell membrane at a very low ratio, whereas large molecules cannot enter cells. Because most materials prepared for therapeutic, preventive or diagnostic purposes, each of which requires an effective amount to be delivered into cells, are large molecules or macromolecules, methods of delivering biologically active macromolecules into cells without damaging the cells both in vivo and ex vivo have been demanded.
As a result of studies conducted to satisfy this demand, protein transduction domains (PTDs) have been suggested, and among them, TAT protein, which is the transcription factor of human immunodeficiency virus-1 (HIV-1), has been most frequently studied. It was found that the TAT protein is more effective in passing through the cell membrane when it is composed of amino acids 47 to 57 (YGRKKRRQRRR), on which positively charged amino acids are concentrated, compared to when it is in a full-length form consisting of 86 amino acids (Fawell, S. et al., Proc. Natl. Acad. Sci. USA, 91:664, 1994). Other examples verifying the effects of PTDs include a peptide having a sequence of amino acids 267 to 300 of the VP22 protein of Herpes Simplex Virus type 1 (HSV-1) (Elliott, G. et al., Cell, 88:223, 1997), a peptide having a sequence of amino acids 84 to 92 of the UL-56 protein of HSV-2 (GeneBank code:D1047[gi:221784]), and a peptide having a sequence of amino acids 339 to 355 of the Antennapedia (ANTP) protein of Drosophila sp (Schwarze, S. R. et al., Trends. Pharmacol. Sci., 21:45, 2000). In addition, artificial peptides consisting of positively charged amino acids also showed the effect of delivering drugs (Laus, R. et al., Nature. Biotechnol., 18:1269, 2000).
Recently, the present inventors reported the preparation of a low-molecular-weight protamine (LMWP) and the cell-penetrating activity thereof, in which the low-molecular-weight protamine (LMWP) has a peptide sequence similar to TAT, serves as a protein transduction domain and contains a large amount of cationic amino acids such as arginine. Particularly, the LMWP peptide is a naturally occurring cationic peptide from protamine and is advantageous in that it presents no toxicity concerns and can be produced in large amounts (Park, Y. J. et al., J. Gene. Med., 700, 2003). Meanwhile, the present inventors have found that the LMWP peptide selectively binds to vascular endothelial growth factor (VEGF) and heparin, which are distributed specifically in tumor tissue, and have expected that the peptide would have the effect of selectively inhibiting tumors. Meanwhile, this LMWP peptide will hereinafter be referred to as “VEGF-binding protein transduction domain (VPTD) or peptide”.
The present inventors have found that a VEGF-binding protein transduction domain (VPTD) peptide represented as SEQ ID NO: 1 or a heparin-binding protein transduction domain (HPTD) peptide represented as SEQ ID NO: 2 binds specifically to vascular endothelial growth factor (VEGF) and heparin in tumor cells or tumor tissues and also accumulates selectively in tumor cells or tumor tissues to inhibit the growth of the tumor cells, thereby completing the present invention.